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Comparative Study
. 2006 Aug;14(8):1293-302.
doi: 10.1016/j.str.2006.06.012.

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2

Martin Renatus  1 , Shirley Gil Parrado, Allan D'Arcy, Ulf Eidhoff, Bernd Gerhartz, Ulrich Hassiepen, Benoit Pierrat, Ralph Riedl, Daniela Vinzenz, Susanne Worpenberg, Markus Kroemer
Affiliations
Comparative Study

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2

Martin Renatus et al. Structure. 2006 Aug.

Abstract

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

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Figures

Figure 1
Figure 1
Structure-Based Sequence Alignment of the Human USP2 and USP7 Catalytic Domains Cα positions that are in a structural alignment within 1.7 Å are highlighted by gray boxes. USP2 residues are numbered according to the corresponding USP7 residues; insertion loops in USP2 relative to USP7 are numbered alphabetically. Active-site residues (catalytic triad: Cys223, His464, Asp/Asn481; and oxyanion hole: Asn218) and the four cysteine residues (Cys334, Cys337, Cys381, Cys384) coordinating a metal ion are shown in bold. USP2 residues poorly defined by electron density are underlined. Secondary structure elements are colored as in Figure 2B (thumb, gray; fingers, lime; palm, blue). Forty-seven USP2 residues that are within 4 Å of ubiquitin are denoted by triangles. Open triangles, interactions with the ubiquitin core (ubiquitin residues 1–71); solid black triangles, interactions with the ubiquitin C terminus (ubiquitin 72–76); solid gray triangles, interactions with both ubiquitin core and C terminus. A consensus sequence, based on the alignment of 54 human USPs (Quesada et al., 2004) and denoted as “all USP,” is included in the alignment (bold capital letters, identical in more than 90% of the sequences; capital letters, identical in more than 80% of the sequences; small letters, identical in more than 60% of the sequences). Regions of high sequence conservation within the human USPs are highlighted with boxes: Cys-box (215–229), QDE-box (292–305), and His-box (446–468, 477–486, and 512–520).
Figure 2
Figure 2
The X-Ray Structure of the USP2 Catalytic Domain in Complex with Ubiquitin The catalytic domain consists of residues 259–605 of full-length USP2. Secondary structure elements are named according to the USP7 structure (Hu et al., 2002). The loops connecting h5 and h5′ as well as s3 and h7 are poorly defined by electron density (see also Figure 1) and are therefore omitted from the final model. Dotted lines highlight these gaps. (A) Overall structure of the USP2/ubiquitin complex (blue/orange ribbon). The active-site residues Asn218, Cys223, His464, and Asn481 as well as the metal binding site Cys334, Cys337, Cys381, and Cys384 are shown as sticks, and the zinc ion is shown as a pink sphere. (B) Overall structure of USP2. Secondary elements forming the palm, the fingers, and the thumb are painted in blue, lime, and gray, respectively. Ubiquitin has been omitted for clarity. (C) Superposition of the catalytic domain of USP2 in blue with the catalytic domain of USP7 in salmon (1BNF). The ubiquitin molecules of both complex structures superimpose perfectly and have been omitted for clarity. (D) Solvent molecules (blue) mediate the binding of ubiquitin (orange) to USP2 (gray surface). (E) Solvent molecules mediating the binding of ubiquitin are shown as blue spheres, and the molecular surface corresponding to USP2 residues within 4.0 Å of the ubiquitin molecule is highlighted in orange. The ubiquitin molecule has been omitted for clarity. (F) Superimposition of buried water molecules (USP2/ubiquitin, blue; USP7/ubiquitin-aldehyde, salmon). Stars highlight equivalent water positions. USP2 is shown as a gray transparent surface. Lys407 of USP2, replacing a water molecule present in the USP7/ubiquitin-aldehyde structure, is shown as sticks.
Figure 3
Figure 3
Substrate Binding Region of USP2 and USP7 (A) USP2/ubiquitin complex: the five C-terminal residues of ubiquitin, Leu-Arg-Leu-Gly-Gly, are shown as orange sticks. USP2 side chains that are within 4.0 Å of the ligand as well as residues Asn218, His464, and Asn481 of the active site are shown as sticks. Putative hydrogen bonds are indicated by yellow dotted lines. (B) Superimposition of the binding region of USP2 (blue) and USP7 (salmon): residues forming the substrate recognition pocket for the ubiquitin C terminus as well as the catalytic Cys223 are shown as sticks. Only USP7 residues that differ from USP2 are labeled.
Figure 4
Figure 4
USP2 Inhibition by Full-Length Ubiquitin and the C-Terminally Truncated Mutants, Ubiquitin 1-74 and 1-73 Residual enzymatic activity was determined at 22°C with ubiquitin-AMC as substrate. The relative activity (vi/vo) is plotted against the inhibitor concentration. From these data, the corresponding Kis were deduced as described in the Experimental Procedures section. The assay was performed in triplicate; the figure shows one representative set of measurements.
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